Method of delivering a protein to poultry

ABSTRACT

A method of delivering a protein to domestic poultry by administering to the poultry by whole body spray an effective amount of a live avirulent derivative of an enteropathogenic bacterium that contains a recombinant gene encoding the protein.

[0001] This is a continuation-in-part of application Ser. No.09/122,299; filed Jul. 24, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to poultry vaccines and, moreparticularly, to a novel method of delivering a protein to poultryinvolving spraying with a live avirulent derivative of anenteropathogenic bacterium.

[0004] 2. Description of Related Art

[0005] Contamination of poultry meat and eggs by enterobacterial humanpathogens, such as Salmonella spp. is a well known cause of illness inhumans when such contaminated products are consumed. The contaminationoccurs predominantly during processing of carcasses after slaughter bycontact with intestinal contents that contain high levels of suchenterobacteria. The enterobacteria colonize the intestinal tract, but donot normally cause disease in the poultry. In order to reduce thecontamination of food with enteropathogens it would thus be desirable todiminish the amount of human enteropathogenic bacteria present in theintestinal tracts of market-age broilers. Efforts to reduce thiscontamination have focused on improved sanitation during production andprocessing (Bailey, J. S., Poult. Sci., 72:1169-1173, 1993), but suchtechniques are time-consuming and expensive and are not totallyeffective in avoiding sporadic contamination. (See, e.g., Food BorneDisease Outlook Annual Summary, 1982; and Salmonella Surveillance AnnualSurvey 1992; both available from Center for Disease Control, U.S.Department of Health and Human Services, Atlanta, Ga.). Methods thatdepend upon sanitation during processing must be repeated frequentlysince processing equipment and personnel can be re-contaminated by eachcontaminated fowl that is processed. Methods that depend upon sanitationduring production require constant vigilance due to the high potentialfor contamination in the production environment. Therefore, a simple andinexpensive method to control enteropathogenic microbes in poultryduring growth would be a key improvement in reducing carcasscontamination during processing.

[0006] Promsopone et al, J. Food Protect., 61(2): 176-180, 1998, havereported that S. typhimurium colonization of the intestinal tracts ofpoultry can be reduced by administration of an avian-specific probioticcombined with S. typhimurium specific antibodies. Lactobacillusacidophilus, Streptococcus faecium and S. typhimurium-specificantibodies were administered by spraying the chicks at one day of agefollowed by oral administration via the drinking water from day 1 to day3. The chicks were challenged by oral administration of S. typhimuriumon day 1 and significantly reduced amounts of S. typhimurium wererecovered from the cecum and colon following probiotic-treatment at 31,38 and 43 days. Although administration of probiotic and antibodies asearly as 1 day of age may have been important in reducing colonizationof the intestine by S. typhimurium, it is not clear from this reportwhether the initial spray administration of probiotic and antibodies orthe more commonly used oral administration in the drinking water on days1-3 was responsible for decreasing S. typhimurium colonization.

[0007] Vaccines for use in preventing diseases in poultry have beenreported and some of these vaccines are specific for Salmonella (See,e.g. U.S. Pat. Nos. 5,294,441, 5,389,368, 5,468,485 and 5,387,744). Themethods for administration of vaccines in poultry vary, however,depending upon the target site of action of the active agent. In fact,it is commonly believed that the vaccination route should be tailoredaccording to the preferential site of the microorganism for localizationand replication. Thus, for Newcastle disease and infectious bronchitisviruses which multiply in the respiratory route, the vaccination methodsof choice would be by eye drop into the eye, nasal passage andrespiratory system of the chick or by the spray route. (Giambrone, WorldPoultry-Misset 13:19-23, 1997). Since many of the more importantdiseases of poultry occur in the respiratory tract, studies reporting onadministration of spray vaccination for these diseases have used sprayadministration because an aerosol or spray is easily inhaled by the birdand thereby contacts the mucosal surfaces of the upper respiratorytract. Administration of vaccines for non-respiratory diseases, such asdiseases of the tissues, circulatory system or gut, is usually bysubcutaneous injection, or by oral administration, either by inoculationor by application in drinking water.

[0008] References disclosing the use of the spray administration ofvaccines to poultry have almost exclusively been directed to immunizingagainst viral agents that invade through the upper respiratory tractsuch as, for example, to prevent Newcastle disease, avianencephalomyelitis, Marek's disease, laryngotracheitis, infectiousbronchitis and the like. The reasons for this state of affairs arelikely based on the commonly accepted knowledge that, in mammals,aerosol immunization has the potential to elicit both mucosal andsystemic immunity, the balance between the two being manipulated by theaerodynamic particle size of the aerosol. Antigen delivered in aerosolswith particle size >5 μm impinges on the mucosa of the upper respiratorytract, is taken up either directly, or delivered to the local lymphatictissue and initiates mucosal immunity. Aerosols <5 μm deposit deep inthe lung where antigen is taken up by lung-associated lymphoid tissueand transported by the draining lymphatics to the bloodstream and thusto the spleen, resulting in both a local and a systemic immune response.(See, Dertzbaugh, M. T., et al., Chap. 52, pp.839-849, at p. 846, inMucosal Immunology, 2nd Ed., P. L. Ogra et al., Eds., Academic Press,San Diego (1999)).

[0009] Bacterial vaccines, in particular live attenuated mutants derivedfrom highly virulent bacterial parent strains, have also been used inpoultry (Roland, K. et aL, Efficacy of Salmonella typhimurium vaccinestrains expressing Escherichia coli 078 lipopolysaccharide to protectagainst E. coli challenge in chickens, Abstract of a presentation atConf. Of Res. Workers in Animal Diseases, Chicago, Ill., Nov. 10, 1997).Derivation of the attenuated mutant strain from a highly virulent parentincreases the likelihood that the attenuated mutant will not onlycolonize the intestinal tract but also colonize the gut associatedlymphoid tissue (GALT) and elicit protective immunity. (See, e.g.,Curtiss Ill et al., in Colonization Control of Human BacterialEnteropathogens in Poultry, Blankenship et al., eds, Academic Press,Inc., New York, 1991, 169-198). In contrast, bacteria that colonize theintestine but do not invade and colonize the GALT may. not elicit animmune reaction. For example, studies in mice have revealed thatlipopolysaccharide (LPS) O-antigen repeats on the surface of S.typhimurium are important not only to withstand nonspecific host defensemechanisms (Microbial Toxins, Vol. V, Roantree et al., eds., AcademicPress, New York, 1971), but also for effective invasion through themucin and glycocalyx covering the intestinal tract. As a consequence,rough mutants lacking LPS O antigens, when given orally, are unable toinvade and colonize the GALT (See, e.g. Curtiss et aL, 1991, supra).

[0010] Some references have reported on the administration of bacterialvaccines to poultry by oral or subcutaneous injection. For example, onecommercial vaccine to prevent paratyphoid in pigeons comprises killed S.typhimurium administered by subcutaneous injection (Vetafarm ParatyphoidVaccine, Vetafarm Pty. Ltd., Wagga Wagga, Australia). In addition,Curtiss et al, 1991, supra, report the use of an avirulent derivative ofa pathogenic Salmonella as an orally administered vaccine in chicks.

[0011] Spray vaccination has also been reported for bacterial vaccinesthat cause respiratory diseases. Hertman et al. report on oral andaerosol administration of a Pasteurella multocida vaccine to chickensand turkeys to prevent fowl cholera, which is a respiratory tractdisease (U.S. Pat. No. 4,169,886). Ley et al. report on eye-drop andaerosol administration of a vaccine containing live Mycoplasmagallisepticum, which produces a respiratory tract disease (Ley et al.,Avian Diseases 41:187-194, 1997). A commercially available vaccinerecommends administration of a vaccine containing an avirulent strain ofE. coli serotype 078 to immunize against the respiratory disease causedby the wild-type parent (see Product Bulletin for GARAVAXS®-T,Schering-Plough Animal Health Corp., Omaha, Nebr.). The use of anaerosol administration for all of these vaccines would have beenselected because the underlying disease for which the poultry were beingvaccinated involved infection of the respiratory tract.

[0012] Another reference reported that a vaccine containing a strain ofthe nonpathogenic E. coli K-12 lacking O-antigen could be administeredas an aerosol (U.S. Pat. No. 4,404,186). Nevertheless, the K-12 strainis a laboratory-adapted strain and is not an enteropathogen and becausethis microbe has no ability to invade and colonize the gut associatedlymphoid tissue, it is likely that any immunity elicited by this vaccinewould have been due to immunization through the respiratory route.

[0013] Localized spraying of bacterial vaccines such as by nasalspraying or ocular spraying had been suggested in some references (forexample, see U.S. Pat. No. 5,294,441). Nevertheless, none of thisearlier work suggested the use of whole body spray administration ofenteropathogenic bacterial vaccines.

[0014] Therefore, while spray-administered vaccines have been reportedto be useful in controlling respiratory diseases in poultry, whole-bodyspray administration has not been suggested for vaccines in poultry forthe control of human pathogens that are often present in and transmittedby poultry, but which are not the causative agents for respiratorydisease in poultry.

[0015] Modem commercial poultry practice sometimes requires the deliveryof proteins to the birds, other than antigenic proteins for the purposeof eliciting an immune response. For example, growth factors,immunoregulatory proteins and peptides, and other biologically activeproteins and peptides are often delivered to birds. The delivery ofthese proteins is most commonly done by inclusion in the feed ordrinking water, but these methods are useless for newly hatched chicks,since they do not eat or drink for up to several days after hatching. Inaddition, the inclusion of labile proteins in drinking water or feed,can often result in the inactivation of a significant portion of theactive molecules due to microbial activity, thermal degradation, oroxidation.

[0016] Accordingly, it would be desirable to provide a method that couldbe used to deliver selected proteins to domestic poultry in a mannerthat would be easy and inexpensive to administer under normal commercialpoultry production conditions, and which would be effective fordelivering the proteins to newly hatched chicks in an effective mannerand without individual handling.

SUMMARY OF THE INVENTION

[0017] In accordance with the present invention, it has been discoveredthat a protein can be administered to domestic birds by whole-bodyspraying of the birds with an effective amount of a live avirulentderivative of an enteropathogenic bacterium that includes a recombinantgene that encodes for the expression of the protein. Theenteropathogenic bacterium that serves as the carrier organism for theprotein may also be referred to herein as a “vaccine” even though thepurpose of the delivered protein or peptide may or may not be to elicitan immune response from the recipient. While virtually any protein canbe delivered by this method, whole-body spray administration issurprisingly effective for proteins that elicit an immune response fromthe host bird, and also for proteins that have some other desirableproperty. The carrier organism for the delivery of the protein is a liveavirulent derivative of an enteropathogenic bacterium. Suchenteropathogenic bacteria are preferably Salmonella species, and it ispreferred that the enteropathogenic bacterium is other than one thatcauses respiratory disease in birds.

[0018] The effective dose, which results in colonization by thebacterium and expression of the protein, is unexpectedly low and isroughly comparable to a dose that is effective by the oral route ofadministration, such as administration in the drinking water. Typically,doses for administration of the live avirulent derivative of theenteropathogenic bacterium of the present invention are from about 10⁵to about 10⁸ colony forming units.

[0019] The spray route of administration of the protein is applicable tobirds, such as chickens, at any age at which they are susceptible to thebeneficial effects of the delivered protein, but is especiallyapplicable to birds that are of an age of 3 weeks or less, and,preferably, to birds of less than 1 day of age.

[0020] In some embodiments, the spray-administration can be followed byadministration of the vaccine in at least one booster dose. Preferablysuch a booster dose can be administered orally by drinking water or byspray at about 14 days after administration by spray.

[0021] Preferably, the spray is a coarse spray of droplets havingdiameters in the range of from about 50 microns to about 150 microns.

[0022] In another embodiment, the invention is directed to a domesticbird to which a protein has been delivered by the method describedabove.

[0023] Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a new method todeliver proteins to domestic poultry in a manner that is easy andinexpensive to administer under normal commercial poultry productionconditions, and which is effective for delivering the proteins to newlyhatched chicks in an effective manner and without individual handling.

BRIEF DESCRIPTION OF THE DRAWINGS:

[0024]FIG. 1 shows the recovery of S. typhimurium χ3985 vaccine strainfrom the spleen and bursal tissues, feces and cecal contents of whiteleghorn chicks 7 days after receiving (a) 10⁵ CFU, (b) 10⁷ CFU, or (c)10⁹ CFU of χ3985 by coarse spray or by direct oral methods of deliveryon day of hatch;

[0025]FIG. 2 shows the recovery of S. typhimurium χ3985 vaccine strainfrom the spleen and bursal tissues, feces and cecal contents of whiteleghorn chicks 20 days after receiving (a) 10⁵ CFU, (b) 10⁷ CFU, or (c)10⁹ CFU of χ3985 by coarse spray or by direct oral methods of deliveryat days 1 and 14; and

[0026]FIG. 3 shows the serum IgM, IgA and IgG responses at 20 days ofage as detected by using purified S. typhimurium LPS in white leghornchickens immunized and boosted with (a) 10⁵ CFU, (b) 10⁷ CFU, or (c) 10⁹CFU of S. typhimurium χ3985 by coarse spray or by direct oral methods ofdelivery at days 1 and 14.

DESCRIPTION OF THE PREFERRED EMBODIMENTS:

[0027] The present invention is based upon the discovery that desirableproteins and/or peptides can be delivered to domestic birds by thewhole-body spray administration of live avirulent derivatives ofenteropathogenic bacteria that encode for the expression of the proteinsand/or peptides.

[0028] The whole-body spray administration method of the presentinvention allows delivery of the vaccine, protein, or immunogeniccompositions to-the gastrointestinal tract of the poultry. Sprayadministration or spray vaccination as used herein is intended to meanthe delivery of droplets of a liquid comprising a live avirulentderivative of an enteropathogenic bacterium that includes a recombinantgene that encodes for the expression of a protein. Whole-body sprayadministration is intended to mean the delivery of such droplets to alarge portion of the entire body of the poultry. This is incontradistinction to a localized spray administration such as is byintranasal spraying in humans in which administration is to only aspecific, small, localized target area. The whole-body spray approachfor administering the carrier organism of the present inventionindiscriminately delivers the microbe to a large portion of the bodysurface of the poultry constituting that portion of the entire bodysurface that is accessible to the spray device (see for example, U.S.Pat. Nos. 4,316,464 and 4,449,968, which are incorporated by reference).Such whole-body spray administration of the organisms of the presentinvention is particularly applicable for administration to large numbersof poultry at the same time.

[0029] Spray administration in the present invention preferably involvesdelivery of a coarse spray containing the vaccine or immunogeniccomposition to the poultry. Although not wishing to be bound by aparticular theory, it is believed that the administration of a vaccineor immunogenic composition as a coarse spray allows the spray dropletsto contact the body surface while minimizing the amount of the vaccinethat is inhaled into the lower respiratory system. This is to bedistinguished from a spray of very fine droplets or mist, such as iscommonly referred to as an aerosol in which droplets have a diameter ofless than about 40 microns. Unlike the aerosol sprays, the coarse sprayof the present invention is believed not to be deeply inhaled whichassists in avoiding the development of respiratory infections seen withsome spray vaccination (See for example, U.S. Pat. No. 4,449,968; Clarkeet al, Austr. Vet. J 56:424-428, 1980). A coarse spray as used herein isintended to mean a spray that is composed of liquid droplets having adiameter sufficient to substantially prevent the inhalation of thedroplets into the lower respiratory system of the bird, but stillcausing the liquid droplets to contact the body surface of the bird. Theconsistency of such a coarse spray has been referred to as “misty rain”,and it is preferred that the spray have less than about 1% of thedroplets in a size range of less than about 12 microns. Preferably, thecoarse spray is composed of droplets having a mean diameter of fromabout 40 to about 400 microns; more preferably from about 40 microns toabout 200 microns, even more preferably from about 50 to about 150microns and most preferably from about 50 to about 100 microns.Alternatively the coarse spray can have about 80% of droplets in a rangeof from about 90 to about 190 microns.

[0030] The type of spray vaccination equipment that is used for theadministration of the vaccine is not critical and almost any type ofspray vaccination equipment capable of dispensing a coarse spray can beused (see for example, U.S. Pat. Nos. 4,316,464, 4,449,968, 4,674 and5,312,353).

[0031] The spray administration of the present invention delivers avaccine comprising a live avirulent derivative of an enteropathogenicbacterium. The vaccine microbe is an enteropathogen that is capable ofcolonizing the intestinal tract and gut associated lymphoid tissues(GALT) of the poultry. Such microbes serve as the immunogenic componentof the vaccine or immunogenic composition and include enteropathogenicbacteria such as Escherichia, Klebsiella, Proteus, Yersinia, andErwinia, Salmonella, Salmonella-Escherichia hybrids, Shigella,Campylobacter, Providencia, Morganella, Hafnia, Serratia, Edwardsiella,Enterobacter and Citrobacter. In particular, Salmonella, Escherichia andSalmonella-Escherichia hybrids are usefuil in the present invention,including, preferably, E. coli and Salmonella such as Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella dublin, Salmonella gallinarum, Salmonellapullorum, Salmonella arizona, Salmonella enteriditis, Salmonellaheidelberg, Salmonella anatum, Salmonella hadar, Salmonella agona,Salmonella Montevideo, Salmonella kentucky, Salmonella infantis,Salmonella schwarzengrund, Salmonella saintpaul, Salmonella brandenburg,Salmonella istanbul, Salmonella cubana, Salmonella bredeney, Salmonellabraenderup, Salmonella livingstone, Salmonella berta, Salmonellacalifornia, Salmonella senftenberg, Salmonella mbandaka and Salmonellacholeraesuis.

[0032] The avirulent derivative of an enteropathogenic bacterium canalso serve as a carrier bacterium to deliver selected antigens to theGALT, gastrointestinal tract, and internal organs. Such carrier bacteriacan contain and express a recombinant gene from a pathogenic organism,for example, so that antibodies and/or cellular immunity will beelicited against the antigenic gene product normally produced by thepathogenic organism. It is thus possible to use the avirulent derivativeof an enteropathogenic bacteria, administered by spray, to deliverantigens to a wide variety of microbes and to elicit an immune responsein the poultry against microbes that need not necessarily be able tocolonize the gastrointestinal (GI) tract.

[0033] The avirulent microbes can additionally be used as vectors forthe synthesis of various proteins in the poultry. Because the avirulentmicrobes of this invention are able to traverse the GALT after sprayadministration and enter into the gastrointestinal tract of the poultry,the microbes can be used to make and deliver heterologous gene productssuch as, for example, growth factors or immunoregulatory products orsubstances that stimulate or suppress various physiological functions.Such microbes contain and express a recombinant gene that encodes thedesired protein.

[0034] The terms enteropathogenic bacterium or enteropathogenic bacteriaare intended to mean microbes that are capable of colonizing theintestinal tract and the gut associated lymphoid system of the poultry.As used herein, “colonizing”, means the enteropathogenic bacteria areable to attach to, invade and persist in one or more of the followingtissues in the vaccinated bird: gut associated lymphoid tissue (GALT),bronchus associated lymphoid tissue (BALT), lung, spleen, liver, bursaof Fabricius, and ceca. As used herein, “pathogen” is intended to mean amicrobe that is capable of causing disease symptoms or impairing normalphysiological fumctioning. The vaccines of the present invention containavirulent derivatives of an enteropathogenic strain of bacteria. Byderivative or derived strain, reference is made to a strain that hasbeen genetically modified from its parent from which it is descended. Bypathogenic it is meant that the microbe is capable of causing disease orimpairing normal physiological functioning.

[0035] Reference to avirulence is intended to mean that a particularvirulent microbe strain has been modified so that it is incapable ofinducing a full suite of symptoms of the disease state that is normallyassociated with its virulent pathogenic counterpart. Thus, avirulenceincludes a state of diminished virulence or ability to produce diseaseconditions and the avirulent microorganisms are not necessarilycompletely absent of any ability to impair normal physiologicalfunctioning of the host. In addition, an avirulent microbe is notnecessarily incapable of ever finctioning as a pathogen, but theparticular microbe being used is avirulent with respect to theparticular individual being treated. Preferably, the enteropathogenicbacterium from which the avirulent microbe is derived is pathogenic atleast to day-of-hatch birds.

[0036] Live avirulent derivatives of enteropathogenic bacteria that havebeen found to be useful in the method of the present invention includethe following:

[0037] Avirulent derivatives as described in U.S. Pat. No. 4,888,170,where the derivatives are of a pathogenic microbe of the genusSalmonella or which is a Salmonella-Escherichia hybrid that express arecombinant gene derived from a pathogen of a vertebrate of the speciesStreptococcus mutans to produce an antigen capable of inducing an immuneresponse in the vertebrate against the pathogen. Preferred strains ofsuch derivatives are S. typhimurium χ3115 (ATCC 39961), and χ3137 (ATCC39962);

[0038] Live avirulent bacterial cells as described in U.S. Pat. No.5,424,065, which are an avirulent Salmonella which contain a mutation inthe phoP gene, wherein the avirulent Salmonella are unable to causeSalmonella-based disease symptoms and are able to colonize in lymphoidtissue for a sufficient time to induce antibody and cellular immunity,and wherein the strain retains the properties of avirulence andimmunogenicity of a Salmonella strain selected from the group consistingof ATCC 53864, ATCC 53865, and ATCC 53866;

[0039] Live avirulent derivatives as described in U.S. Pat. No.5,672,345, where the strains are derivatives of a pathogenic strain ofbacteria characterized by:

[0040] a) a lack of a functioning native chromosomal gene encoding afirst enzyme which is a β-aspartic semialdehyde dehydrogenase (Asd);

[0041] b) the presence of a first recombinant gene encoding a second Asdenzyme wherein the first recombinant gene cannot recombine to replacethe defective chromosomal gene;

[0042] c) the presence of a second recombinant gene encoding a desiredpolypeptide; and

[0043] d) physical linkage between the first recombinant gene and thesecond recombinant gene, wherein loss of the first recombinant genecauses the bacteria to lyse when in an environment which requiresexpression of the first recombinant gene for cell survival. Preferredstrains of such bacteria as are described in U.S. Pat. No. 5,672,345 areprovided by χ6097 (ATCC 67537), χ3520 (ATCC 53681), χ4072 (ATCC 67538),χ3008 (ATCC 53680), χ2108 (ATCC 53678), and χ6097 (ATCC 67813);

[0044] A live avirulent Salmonella, as described in U.S. Pat. No.5,387,744, having a mutation in a cdt gene, where the Salmonella has thephenotype of failure to colonize deep tissue of Salmonella depositstrain ATCC no. 55113. Preferred strains of these bacteria are χ3958(ATCC 55110), χ4323 (ATCC 55115), χ⁴³⁴⁶ (ATCC 55113, χ3940 (ATCC 55119),and χ4073 (ATCC 55118);

[0045] Live avirulent Salmonella choleraesuis as described in U.S. Pat.No. 5,468,485, which strains are obtained from a pathogenic strain of S.choleraesuis, and where the avirulent S. choleraesuis has been madeavirulent by an inactivating mutation in a cya gene and inactivatingmutations in the crp and cdt genes. A preferred strain of such S.choleraesuis is χ3781 (ATCC 67923); and

[0046] Live avirulent Salmonella typhi as described in U.S. Pat. No.5,294,441, where the avirulent S. typhi are obtained from a pathogenicS. typhi strain and are made avirulent by an inactivating mutation inthe structural cya gene and an inactivating mutation in the structuralcrp gene. Useful strains of the type described in U.S. Pat. No.5,294,441 are χ3927 (ATCC 55117) and χ4323 (ATCC 55115), which haveΔcya-12/Δcrp-11 mutations.

[0047] When the subject method of coarse spray administration is used todeliver a protein, to a domestic bird, the terms “deliver a protein” areintended to include the initiation of colonization by the carrierbacterium of at least one of gut-associated lymphoid tissue (GALT),bronchus associated lymphoid tissue (BALT), lung, spleen, liver, bursaof Fabricius, or ceca of the domestic bird, and preferably thecolonization of at least one of the tissues selected from lung, spleen,liver, bursa of Fabricius, or ceca. It is more preferred that, uponcolonization, the carrier bacterium then expresses the recombinant geneto provide the protein it encodes.

[0048] The protein that is delivered by the carrier organism can be aprotein that acts as an antigen to elicit an immune response, asdescribed in detail herein, or it can be a growth factor, regulatoryprotein, or any other protein that it is desirable to introduce into thebird. When the terms “gene product”, or “protein” are used herein in thecontext of their delivery to a bird by the subject method ofadministration, it is to be understood that these terms can pertain to asingle gene product, or protein, but the terms can include two or moregenes and/or proteins, and, in fact, can include a set of genes thatencode enzymes necessary for the synthesis of another desirable proteinor non-protein product.

[0049] By way of example, recombinant carrier bacterium that expressseveral genes are described by Roland et al., in Avian Diseases,43:429-442 (1999), as being live recombinant Salmonella that express anO-antigen of an avian pathogenic gram negative (AP_(G-N)) microbe due tothe stable integration into the Salmonella chromosome of the rfb/rfcgene cluster and the rfc gene of the AP_(G-N) microbe. The Salmonellahost has a mutation in the Salmonella rfb gene cluster or in theSalmonella rfc gene which inactivates the expression of SalmonellaO-antigen, wherein the recombinant Salmonella strain is an attenuated(i.e., avirulent) mutant of a virulent Salmonella strain. Theserecombinant Salmonella can be made to express the E. coli LPS O-antigensof E. coli strains O1, O2, O35 and O78.

[0050] One or more genes encoding immunoregulatory proteins or peptidesmay be recombinantly introduced into the avirulent microbes such thatthe microbes taking up residence in the appropriate immunocompetenttissue are capable of expressing the recombinant product to suppress,augment or modify the immune response in the host. Examples ofimmunoregulatory molecules include but are not limited to: colonystimulating factors (macrophage, granulocyte, or mixed), macrophagechemotoxin, macrophage inhibition factor, leukocyte, inhibitory factors,lymphotoxins, blastogenic factor, interferon, and interleukins.

[0051] One or more genes encoding growth factors can be recombinantlyintroduced into the avirulent microbes, so that when the microbes takeup residence in the appropriate immunocompetent tissue, they are capableof expressing the recombinant product to suppress, augment or modifygrowth-related processes in the host. It is preferred that the growthfactor that is introduced into the host bird is one that is active inthe growth related processes of the host.

[0052] When the protein(s) that are delivered to the domestic bird areantigens and act as vaccines, they elicit an immunological response inthe bird. An immunological response to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, such aresponse consists of the subject producing antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells directedspecifically to an antigen or antigens included in the composition orvaccine of interest.

[0053] By vaccine is meant an agent used to stimulate the immune systemof an individual so that protection is provided against an antigen notrecognized as a self-antigen by the immune system. Immunization refersto the process of inducing a continuing high level of antibody and/orcellular immune response in which T-lymphocytes can either kill theinvading microbe and/or activate other cells (e.g., phagocytes) to do soin an individual, which is directed against a microbe or antigen towhich the organism has been previously exposed. The phrase “immunesystem” is intended to refer to the anatomical features and mechanismsby which an individual produces antibodies against an antigenic materialthat invades the cells of the individual or the extra-cellular fluid ofthe individual and is also intended to include cellular immuneresponses. In the case of antibody production, the antibody so producedcan belong to any of the immunological classes, such as immunoglobulinsA, D, E, G or M. Of particular interest are vaccines which stimulateproduction of immunoglobulin A (IgA) since this is the principleimmunoglobulin produced by the secretory system of warm-blooded animals,although vaccines of the invention are not limited to those whichstimulate IgA production. For example, vaccines of the nature describedherein are likely to produce a broad range of other immune responses inaddition to IgA formation, for example cellular and humoral immunity.Immune responses to-antigens-are well studied and widely reported. Asurvey of immunology is provided in Elgert, Klaus D., Immunology, WileyLiss, Inc., (1996); Stites et al., Basic & Clinical Immunology; 7th Ed.,Appleton & Lange, (1991) the entirety of which are incorporated hereinby reference.

[0054] An individual treated with a vaccine of the present invention isintended to mean one of a species of birds, including domestic birds,particularly those of agricultural importance. Domestic birds or poultryas used herein include any of a variety of domesticated avian species orindividuals of that species, such as chickens, turkeys, ducks, geese,pigeons, guineas, ostriches, emus, and the like and, in particular,those domesticated avian species or individuals kept for the productionof eggs or meat.

[0055] The vaccine can be prepared by growing the vaccine strain insuitable growth media and then used as is or formed into a vaccinecomposition by combining the growing culture, or the cells therefrom,with a suitable diluent. Suitable diluents are preferably liquids andare more preferably a liquid that does not adversely effect thestability and vitality of the vaccine culture and which has a viscositysimilar to water so that it will easily form droplets of a coarse spray.The diluent is preferably free of chlorine, antibiotics, antimicrobials,or any other agent that may be harmful to the live vaccine organisms.Vaccine should be dispersible in the diluent so that no solid lumps orchunks of vaccine remain and the diluent should be at a temperature thatis not harmful to the live vaccine microbes. Examples of suitablediluents include water, distilled water, de-ionized water, skim milk,water containing Marek's vaccine stabilizer, buffered saline withgelatin, and similar compositions that are well-known to persons ofskill in the art. The vaccine is preferably introduced into the diluentwhile the diluent is at a temperature of approximately room temperatureor cooler, more preferably from about 34° C. to about 15° C.

[0056] In one embodiment, vaccine is prepared from S. typhimurium UK-1Δcya Δcrp χ3985 (ATCC 68166). As used herein, this vaccine may bereferred to as χ3985, or as Chi3985, or as χ3985, U.S.D.A. Product Code19C1.01.The vaccine strain can be freshly prepared as described above,or may be recovered from a culture stored, for example, as afreeze-dried culture, in a frozen form (for example, as −70° C. workingseed stock), or otherwise. An inoculum from such culture is then grownto a late log-phase culture in Luria broth in 37° C. By way of example,a −70° C. seed stock can be used to inoculate 50 ml of Luria broth in a250 ml sterile flask covered loosely with foil. The flask is incubatedas a static culture at 37° C. overnight. After about 12-24 hr., 50 ml ofthe static overnight culture is pipetted into 450 ml of prewarmed Luriabroth in a 1 L nonbaffled flask at 37° C. and placed in a New Brunswickincubator shaker at 150 rpm. After the culture reaches OD₆₀₀≧1.0, cellsare pelleted by centrifugation (4400 rpm, 15 min in a Centra MP4centrifuge, IEC swinging bucket 3224 rotor) at room temperature. Cellsare resuspended in 40 ml of room temperature buffered saline withgelatin (BSG). The titer of the vaccine composition can be determined byserially diluting the cell suspension 10-fold in BSG and spreading 100μl of 10⁻⁶ and 10⁻⁷ dilutions onto MacConkey agar +1% maltose forplating. The titer of the vaccine strain is then determined by countingcolonies that develop upon incubation of the plates. The titer isexpressed in terms of colony forming units of the vaccine microbe (CFU)per unit volume of the vaccine composition.

[0057] Vaccine for application to poultry is prepared as described aboveand the culture is diluted to the desired dose density, or titer, in asuitable diluent. The buffer of the diluent, if used, is adjusted tomatch the pH and ionic strength required to maintain the stability andvitality of the vaccine strain. The vaccine is then ready for loadinginto the sprayer and for administration to the poultry.

[0058] Spray administration can also be performed in a manner to delivera particular dosage per bird. One technique that can be used to deliveran accurate vaccine dosage is to spray birds in an enclosed space for acalculated period of time at a known volumetric delivery rate. Byknowing the number of birds to be vaccinated, the desired dosage of thevaccine per bird, the titer of the vaccine and the delivery rate of thespray equipment, one skilled in the art can easily calculate thespraying time required to deliver the required dosage per bird.Furthermore, some models of commercially available spray equipment allowpre-selection of the volume of liquid to be delivered to a known numberof birds.

[0059] The vaccine or immunogenic composition of the present inventionis administered in an effective dose or an effective amount. As usedherein an effective amount is that quantity of vaccine or immunogeniccomposition which is sufficient to elicit an immune response against atarget microbe or antigen for which the poultry is being vaccinated.Such immune response will involve the production of antibodies and/orcellular immunity. In one significant aspect of the present invention,the vaccine or immunogenic composition can be administered at a doseroughly equal to the dose effective upon oral administration, forexample by administration in the drinking water. It is preferred thatthe dose that is administered in the form of a coarse spray is fromabout 10⁴ to about 10⁸ colony forming units of the live avirulentderivative of a pathogenic bacterium; more preferred is a dose of fromabout 10⁵ to about 10⁷ colony forming units; and even more preferred isa dose of at least about 1×10⁶ colony forming units of the liveavirulent derivative of a pathogenic bacterium.

[0060] Preferably the spray administration is given to birds when theyare less than one day old, i.e. on the day of hatch. It is often alsodesirable to administer one or more booster applications of the vaccinesome time after the initial spray administration. Such boosterapplications can be administered at any time during the bird's life atwhich the bird is susceptible to the beneficial effects of the vaccine.Preferably, such booster applications are applied between 5 and 21 daysof age, more preferably between 6 and 15 days of age and still morepreferably between 7 and 14 days of age and most preferably at 7 days ofage or 14 days of age or at both 7 and 14 days of age.

[0061] The booster doses are typically administered orally in thedrinking water although the booster dose can be administered by anyroute including by spray administration. Administration of the vaccinein the drinking water can be performed by any of a number of methodsknown in the art. By way of example only, administration in the drinkingwater can be performed using the following method. First alldisinfectants, sanitizers and antimicrobials are removed from thedrinking water being given to the birds 24 hours prior to vaccineadministration. Such water free of disinfectants, sanitizers andantimicrobials is again given 24 hours after vaccination. The vaccinecan then be mixed in the clean water that contains no sanitizing agentsor antimicrobials. Fifteen liters of vaccine-containing water can beused for 500 birds such as chickens and ample space should be providedfor all birds to drink easily. Water containing vaccine should beconsumed in 2 hours or less. To assure that all birds drink, watershould be withheld for one to two hours prior to administration in thedrinking water.

[0062] Because the dosage amount for spray administration of the vaccineor immunogenic composition is approximately the same as the oral dose inthe drinking water, both dosage amounts are, preferably, about the same.Thus, for example, if 10⁶ colony forming units are administered per birdby spray administration, then, preferably, about 10⁶ colony formingunits are administered per bird in the drinking water. Preferably, theinitial spray administration dose and any subsequent booster dose willdiffer by less than 100 fold, more preferably by less than 10 fold, evenmore preferable by less than three fold and still more preferably byless than 10%.

[0063] It is preferred that the poultry to be vaccinated be of an age atwhich it is susceptible to the beneficial effects of the protein that isdelivered by the carrier bacterium. While this may vary with species, ithas been found that, such beneficial effects are obtained in poultrythat is of an age of from hatching to about 104 weeks of age. It ispreferred that poultry be day-of-hatch to 52 weeks of age, morepreferably from day-of-hatch to 3 weeks of age, even more preferablyday-of-hatch to 2 weeks of age, still more preferably day-of-hatch to 1week of age and most preferably that it be day-of-hatch. As used herein,the phrase “day-of-hatch” may be used interchangeably with the term“less than one day of age”.

[0064] One advantage of the present method is that it is amenable toapplication under conditions that normally occur in commercial poultryraising operations. Typically, large commercial chicken or turkeyraising operations are characterized by large poultry houses having moreor less automated feed and watering systems and housing over 1,000 birdsper house; often over 5,000 birds per house and even over 20,000 birdsper house.

[0065] The present method can be used at the hatchery or at the poultryfarm on newly hatched chicks by spraying the chicks in the chick boxes,or other trays or boxes, prior to their release into the brooder houseor poultry house. Alternatively, either young or older poultry can besprayed after release into the house. (See, e.g., Grieve, Poultry Times,p.18, Sep. 22, 1997; and Giambrone, 1997, supra.)

[0066] Because of the ease of application of the present method, thecost of poultry vaccination can be very low. The high cost of individualchick handling is avoided by the ability to deliver a protein to dozensof chicks at one time and in a matter of seconds. Moreover, the accurateadministration of the dosage of the carrier bacteria to each chickminimizes overdosing and inefficient application of the carrierbacteria.

[0067] The following examples describe preferred embodiments of theinvention. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1

[0068] This example illustrates that spray vaccination of young chickswith a live S. typhimurium vaccine was as effective as direct oraladministration of the vaccine in inducing serum immunity and inproducing colonization of the intestinal tract and visceral tissues bythe vaccine microbe and in inducing serum immunity.

[0069] To determine the efficiency of colonization and induction ofimmunity by a live avirulent Δcya Δcrp mutant S. typhimurium vaccine thepresent study investigated the use of a coarse spray as a means todeliver the primary and booster vaccinations to day-old chicks.Duplicate groups of birds were given the vaccine by the oral route. Inaddition, two smaller groups of birds were given the wild-type S.typhimurium, UK-1 MGN-054s, by the spray and oral inoculation methodsand LD₅₀'s were determined. Colonization of the spleen, bursa,intestinal tract and cecum by the vaccine strain at 7 and 20 days of agewas determined for the groups of birds receiving the vaccine strain bythe coarse spray route and the direct oral method of delivery. Serumantibody responses were measured by ELISA for sera recovered from20-day-old vaccinates.

[0070] Objective:

[0071] The goals of the study were: 1) to determine if young chicksvaccinated by using a coarse spray method of vaccination are asefficiently colonized by the vaccine strain compared to chicksvaccinated by the direct oral route of vaccination, and 2) to evaluateserum immunity elicited by escalating doses of the vaccine given byeither coarse spray of oral administration. Since a lethal endpoint canbe determined in day-old chicks (but not in 3-day-old or older chicks),the wild-type S. typhimurium parent strain was included to determine ifboth methods of delivery could efficiently and comparably cause disease.

[0072] Materials and Methods:

[0073]S. typhimurium UK-1 Δcya-12 Δcrp-11 χ3985 is an attenuated vaccinestrain (see Curtiss III, et al., in Colonization Control of HumanBacterial Enteropathogens in Poultry, Blankenship et al., eds. AcademicPress, New York, 1991, pp. 169-198). This strain was grown as a freshlate log-phase Luria broth culture and subsequently diluted in bufferedsaline with gelatin to the desired dose density. The vaccine wasprepared to deliver escalating doses of about 10⁵, 10⁷ and 10⁹ CFU(colony forming units) to groups of 4 one-day-old white leghorn chicks.The chicks were treated with either coarse spray (droplets ofapproximately 140 micron diameter) using a spray vaccination device(Preval Power Unit, Precision Valve Corporation, Yonkers, N.Y., 10703)or by direct oral vaccination administered with an 18 gauge×7.5 mmfeeding needle with a 3 mm ball attached at the end. Birds weremaintained on Purina Start and Grow without coccidiostats orantibiotics.

[0074] Concurrently, escalating doses of the wild-type S. typhimuriumparent strain MGN-054s were prepared in buffered saline with gelatinfrom a fresh late log-phase Luria broth culture and administered togroups of 3 day-of-hatch white leghorn chicks by either coarse spray orby direct oral delivery.

[0075] All groups of birds that received the vaccine strain onday-of-hatch were administered a booster inoculation on day 14 by thesame route and dosage as used per individual group for the primaryinoculation. At 7 and 20 days of age, groups of birds were euthanizedwith CO₂.asphyxiation and necropsies performed to aseptically recoversamples of the spleen and bursa, fecal matter and cecal contents forenumeration of the vaccine strain.

[0076] Serum was separated from blood collected during the 20-daynecropsy and diluted 1:200 for ELISA. Briefly, purified LPS from S.typhimurium was used as the test antigen. Goat anti-chicken IgA, IgM andIgG affinity-purified polyclonal antibodies EC2-001, EC2-005 andEC2-0011, respectively, (from Immunovision, Springdale, Ark.) were usedat a dilution of 1/5000 to detect chicken IgM, IgA or IgG bound topurified LPS antigen on ELISA plates (Dynatech Laboratories Inc.,Alexandra, Va.). A rabbit anti-goat IgG (whole molecule) alkalinephosphatase conjugate (Sigma, St. Louis, Mo.) was used at a dilution of1/3000 with P-nitrophenyl phosphate (Sigma, St. Louis, Mo.) (1 mg/ml) indiethanolamine buffer pH 9.8 as the substrate. The reaction was stoppedafter 30-min. incubation with 50 μl of 3M sodium hydroxide. Absorbancieswere read at 405 nm using an automated microplate reader (Bio-Tek,Winooski, Vt.). The ELISA results were expressed as mean absorbance (405nm) of four chickens within a treatment group. The cut-off point wastaken as 2× the mean of the absorbance of sera from noninfected chickenswhich served as the baseline for the detection of chicken immunoglobulinisotypes by ELISA.

[0077] Results and Conclusion:

[0078] No mortality was observed in groups of birds that received dosesof the vaccine up to 10⁹ CFU administered by on days 1 and 14. TheLD₅₀'s observed in the groups of day-old chicks that received escalatingdoses of the wild-type virulent parent strain by the coarse spray ororal method of delivery were approximately 10³ CFU, which were the sameas previously determined by oral inoculation in day-old chicks.

[0079]FIG. 1(a, b and c) shows recovery of the vaccine strain from thespleen and bursal tissues and feces and cecal contents of birds 7 daysafter receiving the vaccine strain on day-of-hatch. FIG. 2(a, b and c)shows recovery of the vaccine strain from the spleen and bursal tissuesand feces and cecal contents of birds 20 days after receiving thevaccine strain on days 1 and 14. The vaccine strain colonized visceraland lymphatic tissues as well as the intestinal tract efficiently in100% of the birds sampled. In another study comparing these two methodsof vaccine delivery, similar results were observed with broiler chicksmaintained on Purina Start and Gro supplemented with the recommendedlevels of bacitracin (BMD-50) and a coccidiostat (Coban- 100). For alldose levels compared, the data generally showed no significantdifferences between the use of coarse spray or direct oral inoculationto deliver the S. typhimurium χ3985 vaccine strain to leghorn or broilerbirds.

[0080] The levels of Salmonella-specific IgM, IgA and IgG detected byELISA against S. typhimurium LPS in chickens administered the vaccineχ³⁹⁸⁵ are presented in FIG. 3. Doses ranging from 10⁵ to 10⁹ CFU inducedsignificant humoral antibodies using either delivery method tested. Thehighest dose of 10⁹ CFU induced the greatest antibody response comparedto the lower doses. IgM was the predominant immunoglobulin at 20 days ofage in all birds tested in all dose groups. It has been reported thatIgM is the predominant isotope 3 weeks post-vaccination by the oralroutes. (Hassan and Curtiss, Res. Microbiol., 141:839-850, 1990).Increased antibody responses, which are reflected by higher OD₄₀₅measurements, were observed in the present study in birds that received10⁷ or 10⁹ CFU by spray compared to birds that received 10⁷ or 10⁹ CFUby direct oral method of delivery.

EXAMPLE 2

[0081] This example shows the efficacy of spray vaccination of a liveavirulent S. typhimurium vaccine in preventing colonization of the GItract and intestinal organs by wild-type S. typhimurium.

[0082] Fifty-five one-day-old SPF white leghorn chicks were wing bandedfor use in this trial. At day-of-age, 30 birds were vaccinated by coarsespray with 1×10⁷ CFU per dose of S. typhimurium vaccine carried inapproximately 0.3 ml per bird using a Preval Power Unit spray device,from Precision Valve Corp., Yonkers, N.Y. 10703. The vaccine was χ3985,U.S.D.A. Product Code 19C1.01 (Maine Biological Laboratories, Inc.,Waterville, Me.). Twenty birds received 0.3 ml per bird of distilledwater by coarse spray and served as controls., Five additional birdswere held as contemporary controls. Vaccinates and controls were housedseparately in Horsfal isolation units. At two-weeks of age, thevaccinates were boosted in the drinking water with 1×10⁷ CFU per dose ofthe same vaccine. At six weeks of age all birds in both treatment groupswere individually challenged with an oral dose of 1×10⁶ CFU in 0.1 ml ofwild type S. typhimurium.

[0083] Cloacal swabs were collected from each bird of both treatmentgroups prior to challenge and at the time of sacrifice to monitor forSalmonella sp. Swabs were placed in 5 ml of TBGH broth and incubated for24-36 hours at 42° C. After about 36 hours, the broth was streaked ontobrilliant green agar+35 μg/ml novobiocin (BGAN) and incubated at 42° C.

[0084] At seven weeks of age all birds were euthanized and tissues wererecovered and cultured for the wild type S. typhimurium as follows:Approximately 5 gm each of the spleen, liver, kidney and any organdisplaying visible lesions were aseptically removed from each bird.Spleen, liver and kidney tissues obtained from the same bird werepooled. Any organ displaying visible lesions was collected and processedseparately. In addition, a 10 mm sample of the duodenum, ilea and largeintestine tissues with contents were aseptically obtained from each birdand processed individually. In addition, 1 gram of the ceca withcontents were collected from each bird and processed similarly.

[0085] To culture for Salmonella sp. tissues were placed in a sterileWhirl®Pak bag, macerated in a Stomacher blender, and 5 ml oftetrathionate brilliant green Hajna (TBGH) broth added to each bag. Thetissue bags were incubated for 24-36 hrs at 42° C., following which aloopful from each culture was streaked onto BGAN. Plates were examinedafter 36 hours of incubation at 42° C. for characteristic CFU on BGANagar. After the bag had been incubated for about 48 hours, 1 ml of theTBGH from the bag culture was transferred to a tube of 4 ml of freshTBGH broth, the tube incubated for 5 days, and then streaked onto BGANagar. Plates were examined after 36 hours of incubation at 42° C. Anagglutination test with group B Salmonella antiserum was performed on atleast one colony per plate from all plates to confirm the presence ofthe wild type challenge strain S. typhimurium F98. TABLE 1 Experimentaldesign for vaccination with avirulent S. typhimurium and challenge withwild-type S. typhimurium in chickens. NUMBER AGE AT CHALLENGE OF GROUPTREATMENT* CHALLENGE CULTURE BIRDS 1 Vaccine 6 weeks wild-type 30 S.typhimurium 2 Distilled 6 weeks wild-type 20 water S. typhimurium 3 noneN/A none 5

[0086] Results:

[0087] No clinical reaction to the vaccine was observed after anyvaccination. The vaccine strain was isolated from one of thirty birds 8days after vaccination, but was not recovered from any of the birdsthereafter up to the day of challenge. No deaths occurred during thecourse of the trial.

[0088] Culture results for all pre- and post-challenge cloacal swabs arepresented in Table 2 below. The vaccine organism was isolated on day 8post-vaccination from one of the 30 birds; all vaccinated birds wereculture negative when sampled again on day 42 post-primary vaccination.Seven days after challenge, wild-type S. typhimurium were cultured fromthe cloacal swabs of 13% of the vaccinated birds compared to 40% of thenon-vaccinated birds receiving distilled water only. TABLE 2 Number ofculture positive cloacal swabs sampled from treated and non treatedbirds pre- and post- challenge with wild-type S. typhimurium. PRE- POST-CHALLENGE¹ CHALLENGE² GROUP Treatment Day 8 Day 42 Day 49 1 S.typhimurium 1/30 (3%) 0/30 (0%) 4/30 (13%)³ vaccine 2 Non-treated, 0/20(0%) 0/20 (0%) 8/20 (40%) challenged controls 3 Non-treated, non  0/5(0%)  0/5 (0%)  0/5 (0%) challenged controls

[0089] The vaccinated and non-vaccinated birds also differed in thedegree of colonization of the GI tract and the spleen, liver and kidneyalternatively (see Table 3). Wild-type S. typhimurium were cultured frompooled organ tissues of 85% of the non-vaccinated control birds comparedto 13% of the vaccinated birds (P<0.05). A significant reduction in thenumber of culture positive ileal and large intestine samples was foundfrom vaccinated and non-vaccinated birds (P<0.05). A significantreduction was also found in the number of culture positive cecal samplesfrom vaccinated and non-vaccinated birds (P<0.05). No differences couldbe determined between the numbers of culture positive duodenal samplesfrom the vaccinated and non-vaccinated birds. TABLE 3 Culture of S.typhimurium in broiler age birds vaccinated with avirulent live S.typhimurium challenged with wild-type S. typhimurium. CULTURE RESULTS(No. positive/tested (%)) VACCINATION CHALLENGE Organ Large GROUP (Day:CFU) at 6 weeks (CFU) Pool Duodenum Ilea Intestine Ceca 1 Day 1: 3.6 ×10⁶ 1 × 10⁶  6/30 1/30 (3%)  8/30  5/30  7/30 Day 14: 6.8 × 10⁶ (20%)¹(27%)¹ (17%)¹ (23%)¹ 2 None 1 × 10⁶ 17/20 2/20(10%) 12/20 13/20 10/20(85%) (60%) (65%) (50%) 3 None None  0/5 0/5  0/5  0/5  0/5

[0090] The modified live S. typhimurium vaccine provided protection tovaccinated birds against artificial challenge with invasive wild-type S.typhimurium. This challenge trial showed that birds vaccinated at 1 and14 days of age with the live S. typhimurium vaccine had a statisticallysignificant advantage over control birds. There was a significantreduction in the number of broiler age birds in both treatment groups ininternal organ colonization by the wild-type S. typhimurium (P<0.05).Protection at the gut level was apparent as fewer numbers of vaccinateswere culture positive from ileal, large intestine and cecal samplestested than found from these tissues from non-vaccinated birds (P<0.05).The duodenum does not appear to be a target tissue for S. typhimurium inthat only 10% of the non-vaccinated control birds were colonized by thewild-type. In addition, a significant reduction in the number of birdsshowing positive cloacal culture swabs for the wild-type challengeorganism was noted in the vaccine-treated birds as compared to thenon-treated birds.

[0091] The data from this study demonstrate that S. typhimurium vaccineadministered to chicks on day one as a coarse spray and in drinkingwater at day 14 significantly reduces colonization of the GI tract andinvasion and colonization of visceral organs by wild-type S. typhimuriumin broiler age birds.

EXAMPLE 3

[0092] This example illustrates the efficacy of spray administration ofa live avirulent S. typhimurium vaccine in preventing colonization ofinternal organs following oral challenge with either wild-type S.enteritidis or S. heidelberg.

[0093] Table 4 shows the experimental design of the spray vaccinationstudies in which chickens were challenged with either S. heidelberg orS. enteritidis. TABLE 4 Experimental design for vaccination with S.typhimurium vaccine and challenge with S. heidelberg or S. enteritidisin chickens. AGE AT AGE AT NO. OF GROUP VACCINE VACCINATION CHALLENGECHALLENGE BIRDS 1 Chi3985, Days 1 & 14 S. heidelberg 6 weeks 33 ProductCode 19C1.01 2 None None S. heidelberg 6 weeks 20 3 None None None None10 4 Chi3985, Days 1 & 14 S. enteritidis 6 weeks 33 Product Code 19C1.015 None None S. enteritidis 6 weeks 20 6 None None None None 10

[0094]S. heidelberg Study

[0095] Sixty-three one-day-old SPF white leghorn chicks (SPAFAS Inc.,Storrs, Conn.) were wing banded for use in this trial. At one day ofage, 33 birds (Group 1) were vaccinated by coarse spray with 1×1⁷ CFUper dose of vaccine χ3985, U.S.D.A. Product Code 19C1.01 delivered inapproximately 0.35 ml volume per bird using a Preval Power Unit(Precision Valve Corp., Yonkers, N.Y.) spray device. Twenty birds (Group2) received 0.3 ml per bird of distilled water by coarse spray andserved as controls, ten additional birds (Group 3) were held asnon-vaccinated, non-challenged controls. Vaccinates and controls werehoused separately in Horsfal isolation units.

[0096] At two weeks of age, the vaccinates were boosted in the drinkingwater with 9.6×10⁶ CFU per dose of the same vaccine; for consumption byeach bird in 5.1 ml of water. Serum samples were collected prior tochallenge from six-week-old birds in Groups 1 and 2 to assess Salmonellaantibodies by ELISA. Cloacal swabs were also taken from each bird atthis time to culture for the presence of Salmonella sp. At six weeks ofage, all birds in Groups 1 and 2 were individually challenged with anoral dose of 1.6×10⁸ CFU in 1.0 ml of wild-type nalidixic acid-resistantS. heidelberg.Four days later, all birds were swabbed, euthanized andtissues were recovered and cultured for the wild-type S. heidelbergstrain as follows. Approximately 10 gm each of the spleen,-liver, kidneyand any organ displaying visible lesions were aseptically removed fromeach bird. Spleen, liver and kidney tissues obtained from the same birdwere pooled. Any organ displaying visible lesions was collected andprocessed separately. In addition, a 10 mm sample of the duodenum, ileaand large intestine were aseptically obtained from each bird andprocessed similarly. In addition, 10 mm sample of the ceca with contentsexpressed were collected from each bird and processed similarly.

[0097] To culture for Salmonella sp., tissues were placed in a sterileWhirl®Pak bag, 25 ml of tetrathionate brilliant green Hajna (TBGH) brothadded to each bag and the sample was mascerated in a Stomacher blender.The tissue bags were incubated for 40 hrs at 42° C., following which a10 μl loopful from each culture was streaked onto brilliant green agar+35 μg/ml novobiocin (BGAN) and xylose-lysine-tergitol 4 agar+100 μg/mlnalidixic acid (χLT4-Nal). Plates were examined after 24 hours ofincubation at 42° C. for characteristic colonies on XLT4-Nal and BGANagar. The enrichment broth cultures were incubated for an additional 24hours. The cultures corresponding to negative plates were re-streakedonto XLT4-Nal and BGAN, incubated at 42° C. for 24 hours and observedfor characteristic colonies. An agglutination test with Salmonella Ogroup specific (Group B) antiserum was performed on at least one colonyper plate from all plates to confirm the presence of the wild-typechallenge strain S. heidelberg.

[0098] Cloacal swabs were collected from each bird of all treatmentgroups prior to challenge and at the time of sacrifice to monitor forSalmonella sp. Swabs were placed in 5 ml of TBGH broth and incubated for24-40 hours at 42° C. After about 40 hours, the broth was streaked ontoBGAN and incubated at 42° C. An agglutination test with Salmonella Ogroup specific (Group B) antiserum was performed on at least one colonyper plate from all plates to confirm the presence of the wild-typechallenge strain of S. heidelberg.

[0099] Results:

[0100] No clinical reaction to the vaccine was observed after anyvaccinations. No deaths attributed to the vaccine occurred during thecourse of the trial. One bird in the vaccinated group died from injuryon day 49 of the trial. There were no birds in Group 1 that showed anyvaccine positive cloacal cultures when sampled on day 42post-vaccination.

[0101] Culture results for all pre- and post-challenge cloacal swabs arepresented in Table 5 below. The vaccine organism was not recovered fromany of the birds in Group 1 before challenge. Four days after receivingthe wild-type challenge organism, 50% of the vaccinated birds showedpositive cloacal cultures of the wild-type organism while 70% of thenon-vaccinated control birds were culture positive. These data show nostatistical difference between the two groups in showing positivecloacal cultures for the wild-type organism. TABLE 5 Number of culturepositive cloacal-swab samples from treated and untreated birds pre- andpost-challenge with wild-type S. heidelberg. PRE-CHALLENGE¹POST-CHALLENGE² TREATMENT (Day 42) (Day 46) Group 1: S. 0/33 (0%) 16/32(50%)³ typhimurium vaccine Group 2: Non- 0/20 (0%) 14/20 (70%)³ treated,challenged controls Group 3: Non- 0/10 (0%)  0/10 (0%) treated, non-challenged controls

[0102] Groups of vaccinated and non-treated birds were challenged at 6weeks of age with an oral dose of live wild-type S. heidelberg to assessprotection against internal organ tissue invasion and digestive tractcolonization (See Table 6). A significant difference was found betweenthe vaccinated and non-vaccinated groups challenged with wild-type S.heidelberg; twenty-five percent of the vaccinated birds challenged withwild-type were culture positive in pooled organ tissues compared to 70%of the non-vaccinated control birds (P<0.05). A significant reduction inthe number of culture positive digestive tract pooled samples was foundfrom vaccinated compared to non-vaccinated birds (P<0.05). Nodifferences in the number of culture positive cecal samples were seenbetween the vaccinated and non-vaccinated birds. Non-treated,non-challenged control birds were free of Salmonella sp. TABLE 6Protection of broiler age birds vaccinated with live avirulent S.typhimurium and challenged with wild-type S. heidelberg DIGESTIVE ORGANTRACT GROUP TREATMENT POOL POOL CECA 1 Vaccine +  8/32 (25%)¹ 10/32(31%)¹ 21/32 (66%) challenge 2 Non- 14/20 17/20 (85%) 17/20 (85%)vaccinated (70%) challenged controls 3 Non-  0/10 (0%)  0/10 (0%)  0/10(0%) vaccinated, non- challenged controls

[0103]S. enteritidis Study:

[0104] Table 4 shows the experimental design of the S. enteritidisefficacy study. Sixty-three one-day-old SPF white leghorn chicks (SPAFASInc., Storrs, Conn.) were wing banded for use in this trial. Atday-of-age, 33 birds (Group 4) were vaccinated by coarse spray with1×10⁷ CFU per dose of χ3985, U.S.D.A. Product Code 19C1 .01 vaccinecarried in approximately 0.35 ml volume per bird using a Preval PowerUnit spray device (Precision Valve Corp., Yonkers, N.Y.). Twenty birds(Group 5) received 0.3 ml per bird of distilled water by coarse sprayand served as controls, ten additional birds (Group 6) were held asnon-vaccinated, non-challenged controls. Vaccinates and controls werehoused separately in Horsfal isolation units.

[0105] At two weeks of age, the vaccinates were boosted in the drinkingwater with 9.6×10⁶ CFU per dose of the same vaccine for consumption byeach bird in 5.1 ml of water. Serum samples were collected prior tochallenge from 6-week-old birds in Groups 4, 5 and 6 to assessSalmonella antibodies by ELISA. Cloacal swabs were also taken from eachbird at this time to culture for the presence of Salmonella sp. At sixweeks of age, all birds in Groups 4 and 5 were individually challengedwith an oral dose of 4.5×10⁷ CFU in 1.0 minl of wild-type nalidixicacid-resistant S. enteritidis. Seven days later, all birds were swabbed,euthanized and cultured for the wild-type S. enteritidis strain by themethod described above in the S. heidelberg efficacy study, except thatthe agglutination tests were run with Salmonella O group specific (GroupD) antiserum rather than Group B antiserum.

[0106] Results

[0107] No clinical reaction to the vaccine was observed after anyvaccinations. No deaths attributed to the vaccine occurred during thecourse of the trial. One bird in the vaccine-treated group died,presumably from injury received after heart puncture in an effort tocollect blood for serum antibody evaluation.

[0108] Culture results for all pre- and post-challenge cloacal swabs arepresented in Table 7 below. The vaccine organism was:not-recovered fromany of the birds in Group 4 before challenge. Seven days after receivingthe wild-type challenge organism, 41% of the vaccinated birds showedpositive cloacal cultures for the wild-type organism while 63% of thenon-vaccinated control birds were culture positive. These data show nostatistical difference between the two groups in positive cloacalcultures for the wild-type organism. TABLE 7 Number of culture positivecloacal swabs samples from vaccinated and non-vaccinated birds pre- andpost-challenge with wild-type S. enteritidis. PRE- POST- CHALLENGE¹CHALLENGE² GROUP TREATMENT (Day 42) (Day 49) 4 Vaccine + challenge 0/33(0%) 13/32 (41%)³ 5 Non-vaccinated, 0/20 (0%) 12/19 (63%)³ challengedcontrols 6 Non-vaccinated, 0/10 (0%)  0/10 (0%) non-challenged controls

[0109] The vaccinated and non-vaccinated birds also differed in thedegree of colonization of the GI tract and the spleen, liver and kidneycollectively as shown in Table 8. A significant difference was foundbetween the vaccinated and non-vaccinated groups challenged withwild-type S. enteritidis; nine percent of the vaccinated birdschallenged with wild-type were culture positive in pooled internal organtissues compared to 58% of the non-vaccinated control birds (P<0.05). Asignificant reduction in the number of culture positive digestive tractpooled samples was found from vaccinated and non-vaccinated birds(P<0.05). No differences in the number of culture positive cecal sampleswere seen between the vaccinated and non-vaccinated birds. Non-treated,non-challenged control birds were free of Salmonella sp. TABLE 8Protection of broiler age birds vaccinated with a modified live S.typhimurium vaccine after oral challenge with wild-type S. enteritidis.DIGESTIVE ORGAN TRACT GROUP TREATMENT POOL POOL CECA 4 Vaccine +challenge 3/32 (9%)¹ 4/32 (13%)¹ 16/32 (50%) 5 Non- 11/19 7/19 (37%)11/19 vaccinated, (58%) (58%) challenged controls 6 Non-  0/10 (0%) 0/10(0%)  0/10 (0%) vaccinated, Non- challenged controls

[0110] Serum samples were screened for IgG antibody to S. typhimuriumlipopolysaccharide (LPS) by ELISA. Samples were diluted 1:100 and addedto duplicate wells coated with commercially prepared S. typhimurium LPS.HRP-conjugated rabbit anti-chicken IgG at 1:30,000 was used fordetection. Of the birds vaccinated with S. typhimurium vaccine, 52%developed a strong response to the vaccine with OD₄₉₀ measurementsgreater than 0.3; nine percent responded positively in the mid-range of0.2 -0.3 and 39% measured in the low-range of 0.05-0.1. Sera fromcontrol birds averaged OD₄₉₀ measurements of 0.005 ±0.009, well belowall measurements derived from sera from vaccinated birds.

[0111] Conclusion:

[0112] Day-of-hatch, spray vaccination with the live avirulent S.typhimurium vaccine, χ3985, U.S.D.A. Product Code 19C1.01, followed byoral administration of the vaccine in the drinking water at day 14 didnot produce any adverse reaction in chickens. The vaccine strain was notrecovered from cloacal swabs of spray vaccinated birds when sampled at 6weeks of age or just prior to challenge with the wild-type S.enteritidis or S. heidelberg organisms. All vaccinated birds that weretested mounted a serologic response when exposed to the S. typhimuriumvaccine as compared to non-vaccinated birds.

[0113] The data generated from this trial demonstrate that the S.typhimurium vaccine provided significant protection to reduce the levelof colonization of internal organs by either Salmonella heidelberg or S.enteritidis. Although no differences were seen in the level ofcolonization by the wild-type challenge strains in the ceca ofvaccinated compared to non-vaccinated birds, significant protection fromS. heidelberg or S. enteritidis colonization of the digestive tract wasconferred by vaccination.

EXAMPLE 4

[0114] This shows the efficacy of spray vaccination using a liveavirulent S. typhimurium vaccine in protecting broiler age birds againstinternal organ colonization after challenge with wild-type S.enteritidis.

[0115] Fifty-five one-day-old SPF white leghorn chicks (HyVac, Adel,Iowa) were wing banded for use in this trial. At day-of-age, 30 birds(Group 1) were vaccinated by coarse spray with approximately 1×10⁷ CFUper dose of χ3985, U.S.D.A. Product Code 19C1.01 vaccine (produced byMaine Biological Laboratories, Inc., Waterville, Me.), carried inapproximately 0.3 ml volume per bird using a Preval Power Unit spraydevice (Precision Valve Corp., Yonkers, N.Y.). Twenty birds (Group 2)received 0.3 ml per bird of distilled water by coarse spray and servedas controls, five additional birds (Group 3) were held as contemporarycontrols. Vaccinates and controls were housed separately in Horsfalisolation units. TABLE 9 Experimental design for vaccination with S.typhimurium vaccine and challenged with Salmonella enteritidis. AGE ATAGE AT NO. OF GROUP VACCINE VACCINATION CHALLENGE CHALLENGE BIRDS 1Chi3985, Days 1 & 14 S. enteriditis 6 weeks 30 Product Code 19C1.01 2None None S. enteritidis 6 weeks 20 3 None None None N/A 5

[0116] At two weeks of age, the vaccinates were boosted in the drinkingwater with approximately 1×10⁷ CFU per dose of the same vaccine (onedose in 15 ml of water per bird). At six weeks of age all birds in thevaccine-treated and control groups were individually challenged with anoral dose of 4×10⁷ CFU in 0.5 ml of wild-type S. enteritidis. Cloacalswabs were also collected 5 days post challenge from each bird to assessthe number of wild-type cloacal swabs. All birds were euthanized 5 dayspost challenge, necropsied and tissue samples cultured for the wild typeS. enteritidis as described in Example 2.

[0117] Results:

[0118] No clinical reaction to the vaccine was observed during thevaccination period. No deaths occurred in all treatment groups duringthe course of the trial.

[0119] Groups of vaccinated and non-treated birds were challenged at sixweeks of age with an oral dose of live wild-type S. enteritidis toassess protection against internal organ tissue invasion and GI tractcolonization. (See Table 10). A significant difference was found betweenthe vaccinated and non-vaccinated groups challenged with wild-type S.enteritidis; ninety-five percent of the non-vaccinated control birdschallenged with wild-type were culture positive in pooled organ tissuesafter challenge compared to 20% of the vaccinated birds (P<0.01). Asignificant reduction in the number of culture positive duodenal sampleswas found within the vaccine-treated group and control group (P<0.01).No differences were found between the number of S. enteritidis culturepositive ileal, large intestine or cecal samples from vaccinated andnon-vaccinated birds. TABLE 10 Protection of broiler age birdsvaccinated with a modified live S. typhimurium vaccine after oralchallenge with wild-type S. enteritidis. DIGESTIVE ORGAN LARGE TRACTGROUP TREATMENT POOL DUODENUM ILEA INTESTINE (Pooled data) CECA 1Vaccine  6/30  2/30 (7%)¹ 20/30 26/30 (87%) 27/30 (90%) 29/30 (20%)¹(67%) (97%) 2 Controls 21/22 12/22 (55%) 15/22 22/22 (100%) 22/22 (100%)22/22 (95%) (68%) (100%) 3 Isolated controls  0/5 (0%)  0/5 (0%)  0/5(0%)  0/5 (0%)  0/5 (0%)  0/5 (0%)

[0120] Eighty-six percent of the vaccine treated birds showed positivecloacal cultures for the wild-type challenge organism compared to 100%of-the birds in the non-vaccinated control group.

[0121] Conclusion:

[0122] Day of hatch spray vaccination with the live χ3985, U.S.D.A.Product Code 19C1 .01 vaccine, followed by oral administration of thevaccine in the drinking water at day 14 did not produce any adversereaction in chickens.

[0123] Although protection from wild-type colonization was not apparentin the digestive tract and cecal samples of vaccinated birds,significant protection against internal organ colonization by thewild-type S. enteritidis was demonstrated in the internal organs ofvaccine-treated, challenged birds compared to non-vaccinated, challengedbirds.

EXAMPLE 5

[0124] This example illustrates that spray vaccination of chicks withmodified live S. typhimurium vaccine was safe and efficacious inreducing Salmonella contamination under field conditions of normallarge-scale commercial poultry production.

[0125] Purpose:

[0126] The purpose of this trial was to. evaluate the safety andpotential of the χ3985, U.S.D.A. Product Code 19C1.01 vaccine to reduceSalmonella contamination under normal large scale commercial poultryproduction conditions. The safety of the vaccine was monitored bymeasuring the ability of the product to spread and the survival of thechickens during the grow-out period. The efficacy of the product wasmonitored by bacteriological analyses of post-chill carcass rinses afterprocessing, average weight and the percent-condemnations at processing.

[0127] Materials and Methods:

[0128] Three geographically distinct farms with paired houses werechosen such that each poultry house accommodated a minimum of 16,000birds. This allowed a triple-replicate trial utilizing more than 115,000birds consisting of control birds and birds treated with the modifiedlive S. typhimurium vaccine. An effort was made to identify trialpoultry farms that had a known background history of persistentSalmonella contamination. Table 11 identifies the sites and studyparameters of the three trials. TABLE 11 Description of vaccine fieldsafety trials TIME OF FIELD VACCINE VACCINATION DURATION TRIAL SERIALNO. OF CHICKENS (Days after hatching) OF TRIAL SITE USED VACCINATEDCONTROL FIRST BOOSTER (DAYS) 1 7002 21,000 21,000 1 14 48 2 7003 20,50020,500 1 17 42 3 7004 16,000 16,000 1 15 64

[0129] Production of Prelicensing Serials:

[0130] The modified live χ3985, U.S.D.A. Product Code 19C1.01 vaccineprelicensing serials were manufactured following the Outline ofProduction filed and approved Mar. 20, 1995.

[0131] Trial Procedure:

[0132] At all farm sites, the normal commercial vaccination, feeding andwatering regimens of that particular poultry establishment werefollowed. Hatchlings received Marek's vaccine either in ovo or at day ofhatch and Newcastle/bronchitis vaccine by spray at day of hatch. Abooster bronchitis vaccination was administered in both houses on eachfarm site on day 16-18 of the study.

[0133] Application Procedures for the S. typhimurium Vaccine:

[0134] 1. Spray Application at Day of Age:

[0135] A portable spray box apparatus (supplied and operated by MerialSelect, Atlanta, Ga.) was set up in the hatchery so that incoming boxesof birds were vaccinated prior to setting the chicks out on the floor ofthe house. Vaccine was supplied as a lyophilized formulation in a glassvial. The vaccine was mixed into clean, non-chlorinated water accordingto the package instructions. The spray equipment was pre-calibratedmanually to deliver approximately 700 ml to 1 liter over 10,000 chicksunder a pressure of 40-50 psi using 4 coarse anvil fantail spray nozzlesto deliver 50-100 micron sized droplets. This delivered approximately0.07 -0.1 ml per chick.

[0136] 2. Drinking Water Application at Two Weeks of Age:

[0137] Two-week old birds were deprived of drinking water for a periodof two to four hours before allowing birds in the treated house on eachfarm access to the vaccine water. The Merial Select Bag Boost system wasimplemented for calibrated delivery of the vaccine. The requiredquantity of the S. typhimurium vaccine was mixed according to thepackage instructions to a final volume of diluent sufficient toadminister one dose per bird over a two hour period through all housewater lines. This vaccine water was the bird's only source of water overthis two-hour period to ensure that all birds had the opportunity todrink sufficient vaccine. The well water used in the houses on all farmswas tested prior to water vaccination and was found to be negative forchlorine residue and within a pH range of 6.0-7.0.

[0138] Sampling and Enrichment Culture Procedures During the Trial:

[0139] To monitor the presence of indigenous wild-type Salmonella spp.in the environment and in the chick, base-line samples of meconia fromchick papers were collected in the hatchery, while litter drag swabs,feed and water were collected in the houses for bacteriological analysisas described below. Drag swabs of the litter, feed and water sampleswere collected periodically throughout the trials at each test site..

[0140] 1. Chick Papers and Swabs:

[0141] A random sample of 12-18 chick papers and swabs representative ofeach breeder flock was collected from individual chick transport boxesbefore administering the vaccine. Each chick paper selected containingmeconia was placed in individual plastic bags, which were sealed andlabeled. Sterile gauze pads wetted with sterile skim milk were used toswab additional chick papers, then placed in plastic Whirl® Pak bags,sealed and labeled. All samples were immediately transported on icepacks to the laboratory for analysis of Salmonella spp. to determine abase-line level of contamination of incoming chicks.

[0142] 2. Feed and Water Samples:

[0143] Before the start of the study, a 40 g sample of feed was takenfrom the internal bin auger inflow stream from each farm. These sampleswere placed in Whirl® Pak bags and labeled. In addition, 100 ml of waterwas collected at the source after the filter-and 100 ml of watercollected from the nipple drinker from each house. The collected waterwas immediately analyzed for chlorine residue and range of pH. Samplesof feed and water were collected every two weeks throughout the trialperiod for each farm, labeled and transported on ice packs to thelaboratory for bacteriological analysis.

[0144] 3. Drag Swab Samples of House Litter:

[0145] Before the start of each trial, drag swab samples of the litterwere collected from each house on each farm to determine base-linelevels of Salmonella contamination. Additional drag swab samples of eachhouse on each farm were collected every two weeks throughout the trialperiod. Drag swab assemblies were constructed following the NationalPoultry Improvement Plan and Auxiliary Provisions (National PoultryImprovement Plan and Auxiliary Provisions manual. United StatesDepartment of Agriculture, Animal and Plant Health Inspection Service(APHIS 92-55-017), April 1993). The bags were sealed, labeled andtransported on ice packs to the laboratory for bacteriological analyses.

[0146] 4. Culture Procedures:

[0147] Buffered peptone water (International Bioproducts Inc., Redman,Wash.; Difco Inc., Detroit, Mich.) was used to pre-enrich feed, water,chick swabs, chick papers and drag swabs and served as the rinse toassess the bacterial load on carcasses. Tetrathionate brilliantgreen-Hajna broth (Northeast Laboratories Inc., Waterville, Me.) wasused as selective media for enrichment of Salmonella sp. or the vaccine.Tetrathionate brilliant green agar supplemented with 35 μg novobiocin/ml(Sigma, St. Louis, Mo.) was used to identify characteristic growth ofSalmonella sp. or the vaccine. Confirmation of Salmonella wasaccomplished following the procedures outlined in the FSIS SampleCollection Guidelines and Procedure for Isolation and Identification ofSalmonella from Raw Meat and Poultry Products. All positive cultureswere sent to National Veterinary Services Laboratory (Ames, Iowa.) forserotyping.

[0148] 5. Bacteriological Assessment of Whole Carcasses:

[0149] Fifty carcasses were randomly selected post-chill afterprocessing from each house. The FSIS Sample Collection Guidelines andProcedure for Isolation and Identification of Salmonella from Raw Meatand Poultry Products (Food Safety Inspection Service Sample CollectionGuidelines and Procedure for Isolation and Identification of Salmonellafrom Raw Meat and Poultry Products, Federal Register Vol. 61, No. 144,7/25/96, page 38923), was followed to collect whole carcass rinses andto assess the presence or absence of Salmonella sp. and/or vaccineresidues. In addition, PCR (BACS System, Qaulicom, Wilmington, Del.) wasused to verify samples that were positive for agglutination by antiserato specific Salmonella O-antigen groups (Difco Inc., Detroit, Mich.),but where pure cultures could not be obtained.

[0150] Statistical Methods:

[0151] The χ-Square test was used to compare parametric results betweenvaccine-treated and control houses.

[0152] Results and Discussion:

[0153] Trial at Site No. 1:

[0154] 1. Analyses of Base-Line Samples:

[0155] Analyses of base-line samples consisting of chick papers andpaper swabs, water, feed and drag swabs of the house litter collectedprior to the start of the trial revealed that the feed from the controlhouse was positive for Salmonella mbandaka and chicks from two of threebreeder flocks showed positive cloacal cultures for Salmonellaheidelberg. Chicks from one of the breeder flocks showed positivecloacal cultures for organisms suspected to be of O-antigen group C₃,however a pure isolate could not be recovered. No other Salmonella sp.or vaccine organisms could be recovered from feed, water or from dragswabs of the litter from either house on the farm for the remainder ofthe 6-week grow-out period of the trial.

[0156] 2. Livability:

[0157] Livability data were collected weekly by the grower for eachhouse on the farm. Table 12 shows the numbers of birds that expiredduring the grow-out period of the trial. No difference was seen in theloss of birds between the control and treated house where the percenttotal loss for both houses was 2.8%, respectively. The average percentmortality for a period coinciding with the trial for the region of thecountry in which Site #1 was located was 4.4-4.7% (The Poultry InformedProfessional, Department of Avian Medicine, University of Georgia,Athens, Ga., January, 1998). TABLE 12 Mortality of birds during thegrow-out period for Site #1 trial. CONTROL TREATED 1st Week 205 210  2ndWeek 117 87 3rd Week 57 55 4th Week 62 63 5th Week 74 81 6th Week 81 87Total mortality 596  583¹  headcount Percent total 2.8% 2.8%

[0158] The historical livability percentile for Site #1 ranged from 95.5to 99.6 in 1996-1997. The livability percentile of 97.2 for both thecontrol and treated birds was within this range. Based on these data,the vaccine did not have any adverse effect on the livability of thebirds.

[0159] 3. FSIS Inspector's Condemnation Report at Processing:

[0160] The USDA Inspector's condemnation report for each house ispresented in Table 13. The Inspector's report indicated an increase ofairsacculitis and inflammatory process (IP) in the carcasses from thetreated house over the control house. Average values for the region wereobtained from The Poultry Informed Professional—for a week during thetrial. TABLE 13 Percent condemnations at processing for Site #1 trial.CONDEMNATION AVERAGE % CAUSE CONTROLS TREATED FOR REGION Leukosis 0.020.02 0.03 Septicemia/Toxemia 0.25 0.28 0.35 Tumors 0.28 0.15 N/A¹Airsacculitis 0.14 0.51 0.17 Synovitis 0 0 N/A Inflammatory process 0.642.06 0.14 Overscald 0 0 N/A Bruises <0.01 <0.01 0.02 Tuberculosis 0 0N/A Cadavers <0.01 <0.01 N/A Contaminated <0.01 <0.01 N/A Dead onarrival 0.3 0.24 N/A Total condemnations 1.33 3.02²

[0161] The historical data provided by the collaborator shows that thistest farm had experienced cyclic episodes of respiratory disease in thepast two years as airsacculitis percentiles had been elevated during thewinter months. The flock that was on the farm prior to this vaccinatedtest flock showed airsacculitis percentiles of 1.71, compared to 0.51for the birds in the treated house from this trial. There are nodocumented cases of Salmonella spp. causing airsacculitis orinflammatory process. In addition, the carcass inspection process wasnot blinded as the USDA Inspector in Charge required identification ofthe treated birds. Airsacculitis and IP percentiles were not differentbetween controls and vaccine-treated birds in two subsequent trialsdescribed in this report. Based on these reasons, an assessment cannotbe made for the cause of these conditions as attributed to use of thevaccine product.

[0162] 4. Evaluation of the Carcass Weight at Processing.

[0163] The average weight of the bird at processing is an indicator ofperformance of the bird during grow-out period. An examination of eightgrow-out cycles for Site #1 showed average bird weights ranging from4.36 to 4.79 lbs for birds up to 49 days of age; the treated birdweights fell within this range averaging 4.84 lbs compared to 4.85 lbsfor control birds at 48 days of age. Based on these average weight data,the vaccine did not affect the ability of the birds to maintain a levelof performance expected by the producer.

[0164] 5. Carcass Rinse Evaluation.

[0165] The results of carcass rinse evaluation are shown in Table 14.The number of Salmonella positive samples as identified by PCR andO-antigen antisera agglutination from carcass rinses was significantlyless in the treatment group than that found in the control group(p:≦0.05). Neither the modified live S. typhimuriumorganisms nor anyindigenous Salmonella sp. was identified from the 50 carcass rinsesamples from the treated group analyzed in the laboratory. However,eight percent of the rinse samples taken from the 50 carcasses from thecontrol group were positive for Group C Salmonella indicating that thevaccine was efficacious in eliminating indigenous Salmonella from themeat product. TABLE 14 Site #1 carcass rinse evaluation. NUMBER OFPOSITIVE GROUP SAMPLES Control 4/50 (8%) Treated 0/50 (0%)¹

[0166]1. Analyses of Base-line Samples.

[0167] Analyses of base-line samples collected revealed that the feedfrom the control house and the meconia from chick papers containedorganisms suspected to be of O-antigen group C₃, however pure isolateswere not recovered. No other Salmonella sp. or vaccine organism could beidentified from feed, water or from drag swabs of the litter from eitherhouse on the farm for the remainder of the 6-week grow-out period of thetrial.

[0168] 2. Livability.

[0169] Livability data were collected periodically for each house on thefarm. Table 15 shows the mortality data during the grow-out period ofthe trial. Fewer birds expired in the control house than the treatedhouse during the first week of the trial. No differences in the numberof birds that expired between the groups were observed after the firstweek of the trial. The average percent mortality for a week during thetrial period was 5.0-6.2 for the region of the country in which thetrial took place (The Poultry Informed Professional, February 1998). Themortality in each house fell below the regional average for this period.TABLE 15 Mortality of birds during the grow-out period for the Site #2trial. CONTROLS TREATED Day 7 236 363  Day 14 88 73 Day 30 281 306  Day35 64 60 Day 42 82 78 Total mortality headcount 751  880¹  Percent total3.7   4.3

[0170] The 0.6 percent difference in mortality between the treated andcontrol birds in the first week is within the expected deviationobserved for hatchlings. There was no difference in the numbers ofmortality headcount after the first week through the end of the trial.This difference in first week mortality was not observed in two of thethree trials conducted with the vaccine product. Factors that can affectsurvival of hatchlings can include variations of the age of the breederchickens, the quality of the breeder and hatchery management andextended exposure to temperatures less than 85° F. Survival percentilesfor 40-42 day old birds previously raised on this test farm during thesame year as the trial ranged from 94.7 to 97.5. The survival percentilefor the control and treated birds fell within this range with 96.3 and95.7 percent, respectively.

[0171] 3. FSIS Inspector's Condemnation Report at Processing.

[0172] The USDA Inspector's condemnation report for each house ispresented in Table 16. Data for the average %.for the same region of thecountry for site #2 was obtained from the Poultry Informed Professional,February, 1998. No difference in the number of birds condemned in thecontrol and treated groups was observed in this trial. TABLE 16 Percentcondemnations at Site #2 trial. AVERAGE % FOR CONDEMNATION SAME REGIONAS CAUSE CONTROL TREATED SITE #2 Leukosis <0.01 <0.01  0.01Septicemia/Toxemia 0.21 0.13 0.41 Tumors 0.07 0.02 N/A¹ Airsacculitis0.25 0.18 0.39 Ascites 0.11 0.15 N/A Inflammatory process 0.43 0.58 0.45Overscald 0.05 0.01 N/A Bruises 0.03 0.04 0.08 Tuberculosis 0   0   N/ACadavers 0.03 0.04 N/A Contaminated 0.05 0.05 N/A Dead on arrival 0.210.21 N/A Total condemnations 1.44  1.41²

[0173] 4. Evaluation of Carcass Weight at Processing.

[0174] The average weight of birds during nine grow-out cycles on Site#2 ranged from 3.35 to 3.93 lbs; the treated bird weights fell withinthis range averaging 3.7 lbs at 42 days of age. The weight of the birdsfrom the treated house were 6.1% lower than the control birds' averageweight at processing. However, the weight of the treated birds cannot becompared to the control group as two of the four water lines in thetreated house were discovered to be blocked during the fourth week ofthe trial. It was the opinion of the Broiler Manager that this weightdifference may be partially or wholly due to this problem.

[0175] 5. Carcass Rinse Evaluation.

[0176] Table 17 shows Salmonella positive samples identified fromcarcass rinses in treatment group. No vaccine organisms or wild-typeSalmonella sp. were identified in any of the 100 rinse samples fromeither group. TABLE 17 Site #2 carcass rinse evaluation. NUMBER OFPOSITIVE GROUP SAMPLES Controls 0/50 Treated 0/50

[0177] Trial at Site #3:

[0178] 1. Analyses of Base-line Samples.

[0179] Analyses of base-line samples collected revealed that the litterdrag swabs taken from the control house contained organisms suspected tobe of O-antigen Group C₃, however a pure isolate could not be recovered.The chicks originating from two breeder flocks were culture negative forindigenous Salmonella spp. No other Salmonella spp. or vaccine organismcould be identified from feed, water or from drag swabs of the litterfrom either house on the farm for the remainder of the 64-day grow-outperiod of the trial.

[0180] 2. Livability.

[0181] Livabality data were collected weekly for each house. Table 18shows the mortatilty data collected during the grow-out period of thetrial. TABLE 18 Mortality of birds during the grow-out period for Site#3 trial. CONTROLS TREATED 1st Week 293  328 2nd Week 151  176 3rd Week76  114 4th Week 60  70 5th Week 86  68 6th Week 103  97 7th Week 109 84 8th Week 97  118 9th Week 105  119 Total mortality headcount 10801174¹ Percent total 6.8   7.3

[0182] It should be noted that a difference in mortality between thecontrol and treated groups was due to only a five bird deviation. It isour speculation that a standard deviation would exist due to thesignificant number of errors discovered on the daily mortality charts.Therefore, given the uncertainty of the actual headcounts one couldexpect a margin of error as low as 0.03% to contribute significantly tothe final statistical differences. However, when the final survivalvalues were compared for the previous six-months grow-out cycles forthis site for the year of the trial, percentiles ranged from 90 to 95.The livability percentiles for control and treated birds from this trialwere 93.2 and 92.7, respectively, and fell within this range.

[0183] 3. FSIS Inspector's Condemnation Report at Processing.

[0184] Condemnation data for the two treatment groups from Site #3carcasses was inadvertently combined by the USDA Inspectors. TheInspector's condemnation report for both houses is presented in Table19. Averages for the region including Site #3 are based on a 6-week oldbroiler for the same period as the trial (The Poultry InformedProfessional, April, 1998). The condemnation rates for the present trialin the five categories specified in Table 19 are the same or lower thanthose for the region of the country in which Site #3 was located asmeasured for a week during the trial period. The treatment of the birdswith the vaccine did not adversely affect the condemnation percentilesin any category inspected. TABLE 19 Percent condemnations at processingfor Site #3. COMBINED DATA (%) FOR AVERAGE (%) CONDEMNATION TREATED ANDFOR SAME CAUSE CONTROL BIRDS REGION Leukosis <0.01 0.04Septicemia/Toxemia 0.26 0.26 Tumors 0.01 N/A¹ Airsacculitis 0.16 0.3Ascites 0.05 N/A Inflammatory process 0.1 0.3 Overscald 0 N/A Bruises 00.01 Tuberculosis 0 N/A Cadavers 0.04 N/A Contaminated <0.01 N/A Dead onarrival 0.3 N/A Total condemnations 0.92

[0185] 4. Evaluation of Carcass Weight at Processing.

[0186] An examination of three previous grow-out cycles for Site #3showed birds' weights ranging from 6.67 to 7.2 lbs. For this study, theaverage weight of the birds at processing was 7.33 lbs. It should benoted that although this producer has experienced over the past year adecline in the quality of the chicks received from the breeder (as notedby the high mortality during the first two weeks of the trial), theexcellent performance and final heavy weight of the birds at processingfor this trial was unexpected. Based on these average weight data, thevaccine did not affect the ability of the birds to maintain a level ofperformance expected by the producer.

[0187] 5. Carcass Rinse Evaluation.

[0188] Table 20 shows the results of the analyses of carcass rinsesamples for each group. Neither the modified live S. typhimuriumorganisms nor any indigenous Salmonella sp. was recovered from the 50treated house carcass rinse samples. However, twelve percent of therinse samples taken from the 50 carcasses from the control group werePCR and culture positive for S. heidelberg and S. hadar. TABLE 20 Site#3 carcass rinse evaluation. NUMBER OF POSITIVE GROUP SAMPLES Control6/50 (12%)¹ Treated 0/50 (0%)²

[0189] Conclusions:

[0190] Spray vaccination with the live S. typhimurium vaccine, χ3985,U.S.D.A. Product Code 19C1.01, was found to be safe for use incommercial broiler chickens. The birds maintained a level of health andperformance expected by the producer after exposure to the vaccineproduct. The immediate test environment was found to be free of vaccineresidue. The vaccine was found to be efficacious in that its useresulted in complete elimination of indigenous Salmonella sp. on thecarcasses from the treated groups compared to the control groups in twoof three trials. These results confirm that the vaccine was found to beboth safe and efficacious in trials conducted in cooperation with threecommercial poultry operations.

[0191] All references cited in this specification, including withoutlimitation all patents, patent applications, reports, articles fromjournals, periodicals, textbooks and the like, manuscripts, internetwebsite listings, brochures, and any and all other publications, arehereby incorporated by reference. The discussion of the referencesherein is intended merely to summarize the assertions made by theirauthors and no admission is made that any reference constitutes priorart. Applicants reserve the right to challenge the accuracy andpertinency of the cited references.

[0192] In view of the above, it will be seen that the several advantagesof the invention are achieved and other advantageous results obtained.

[0193] As various changes could be made in the above methods andcompositions without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

What is claimed is:
 1. A method of delivering a protein to a domesticbird comprising administering to the bird in a whole-body spray aneffective amount of a live avirulent derivative of an enteropathogenicbacterium comprising a recombinant gene that encodes for the expressionof the protein, wherein the enteropathogenic bacterium is other than onethat causes respiratory disease in birds.
 2. The method according toclaim 1 wherein the method comprises colonization by theenteropathogenic bacteria of the tissues of at least one of the gutassociated lymphoid tissue (GALT), bronchus associate lymphoid tissue(BALT), lung, liver, spleen, bursa of Fabricius, and ceca of thedomestic bird.
 3. The method according to claim 2 where the colonizationfurther comprises expression of the protein encoded by the recombinantgene.
 4. The method according to claim 1 wherein the enteropathogenicbacterium is selected from the group consisting of Escherichia,Klebsiella, Proteus, Yersinia, and Erwinia, Salmonella,Salmonella-Escherichia hybrids, Shigella, Campylobacter, Providencia,Morganella, Hafnia, Serratia, Edwardsiella, Enterobacter andCitrobacter.
 5. The method according to claim 4 wherein theenteropathogenic bacterium is selected from the group consisting ofSalmonella, Escherichia and Salmonella-Escherichia hybrids.
 6. Themethod according to claim 5 wherein the enteropathogenic bacterium isselected from the group consisting of Escherichia coli, Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella dublin, Salmonella gallinarum, Salmonellapullorum, Salmonella arizona, Salmonella enteriditis, Salmonellaheidelberg, Salmonella anatum, Salmonella hadar, Salmonella agona,Salmonella montevideo, Salmonella kentucky, Salmonella infantis,Salmonella schwarzengrund, Salmonella saintpaul, Salmonella brandenburg,Salmonella istanbul, Salmonella cubana, Salmonella bredeney, Salmonellabraenderup, Salmonella livingstone, Salmonella berta, Salmonellacalifornia, Salmonella senftenberg, Salmonella mbandaka and Salmonellacholeraesuis.
 7. The method according to claim 6 wherein theenteropathogenic bacterium is selected from the group consisting ofSalmonella typhimurium strains χ3115 (ATCC 39961), and χ3137 (ATCC39962).
 8. The method according to claim 5 wherein the enteropathogenicbacteria are an avirulent Salmonella which contains a mutation in thephoP gene, wherein the avirulent Salmonella is unable to causeSalmonella-based disease symptoms and is able to colonize in lymphoidtissue for a sufficient time to induce antibody and cellular immunity,and wherein the strain retains the properties of avirulence andimmunogenicity of a Salmonella strain selected from the group consistingof ATCC 53864, ATCC 53865, and ATCC
 53866. 9. The method according toclaim 1 wherein the enteropathogenic bacteria are a derivative of apathogenic strain of bacteria characterized by: a) a lack of afunctioning native chromosomal gene encoding a first enzyme which is aβ-aspartic semialdehyde dehydrogenase (Asd); b) the presence of a firstrecombinant gene encoding a second Asd enzyme wherein the firstrecombinant gene cannot recombine to replace the defective chromosomalgene; c) the presence of a second recombinant gene encoding a desiredpolypeptide; and d) physical linkage between the first recombinant geneand the second recombinant gene, wherein loss of the first recombinantgene causes the bacteria to lyse when in an environment which requiresexpression of the first recombinant gene for cell survival.
 10. Themethod according to claim 9 wherein the enteropathogenic bacteria areselected from the group of strains consisting of χ6097 (ATCC 67537),χ3520 (ATCC 53681), χ4072 (ATCC 67538), χ3008 (ATCC 53680), χ2108 (ATCC53678), and χ6097 (ATCC 67813).
 11. The method according to claim 5wherein the enteropathogenic bacterium comprises a live avirulentSalmonella, having a mutation in a cdt gene, where the Salmonella hasthe phenotype of failure to colonize deep tissue of Salmonella depositstrain ATCC no.
 55113. 12. The method according to claim 11 wherein theenteropathogenic bacterium is selected from the group of strainsconsisting of χ3958 (ATCC 55110), χ4323 (ATCC 55115), χ4346 (ATCC 55113,χ3940 (ATCC 55119), and χ4073 (ATCC 55118).
 13. The method according toclaim 6 wherein the enteropathogenic bacterium is a live avirulentSalmonella choleraesuis obtained from a pathogenic strain of S.choleraesuis, and where the avirulent S. choleraesuis has been madeavirulent by an inactivating mutation in a cya gene and an inactivatingmutation in a crp gene.
 14. The method according to claim 13 wherein theenteropathogenic bacterium comprises strain χ3781 (ATCC 67923).
 15. Themethod according to claim 6 wherein the enteropathogenic bacterium is alive avirulent Salmonella typhi that is obtained from a pathogenic S.typhi strain and is made avirulent by an inactivating mutation in thestructural cya gene and an inactivating mutation in the structural crpgene.
 16. The method according to claim 15 wherein the enteropathogenicbacteria are selected from the group of strains consisting of χ3927(ATCC 55117) and χ4323 (ATCC 55115).
 17. The method according to claim 4wherein the live avirulent derivative of an enteropathogenic bacteriumis a Salmonella and the protein comprises an O-antigen of an avianpathogenic gram negative microbe (AP_(G-N)), where the O-antigen isencoded by an rfb/rfc gene cluster of the AP_(G-N) microbe that isstably integrated into the Salmonella chromosome and having a mutationin the Salmonella rfb gene cluster or in the Salmonella rfc gene whichinactivates expression of Salmonella O-antigen, wherein the recombinantSalmonella strain is an attenuated mutant of a virulent Salmonellastrain.
 18. The method according to claim 17 wherein the O-antigencomprises at least one E. coli specific antigen selected from the groupconsisting of LPS O-antigen of strains O1, O2, O35 and O78.
 19. Themethod according to claim 4 wherein the protein is selected from thegroup consisting of immunoregulatory peptides, immunoregulatory proteinsand growth factors.
 20. The method according to claim 19 wherein theimmunoregulatory peptide or protein is selected from the groupconsisting of macrophage colony stimulating factors, granulocyte colonystimulating factors, mixed colony stimulating factors, macrophagechemotoxins, macrophage inhibition factors, leukocytes, inhibitoryfactors, lymphotoxins, blastogenic factors, interferon, andinterleukins.
 21. The method according to claim 19 wherein the proteinis a growth factor.
 22. The method according to claim 1 wherein thespray administration comprises spraying droplets having diameters in therange of from 50 microns to 150 microns.
 23. The method according toclaim 22 wherein the spray is administered in a dose of from about 10⁴to about 10⁸ colony forming units of the live avirulent derivative of apathogenic bacterium.
 24. The method according to claim 23 wherein thespray is administered in a dose of from about 10⁵ to about 10⁷ colonyforming units of the live avirulent derivative of a pathogenicbacterium.
 25. The method according to claim 24 wherein the spray isadministered in a dose of at least about 1×10⁶ colony forming units ofthe live avirulent derivative of a pathogenic bacterium.
 26. The methodaccording to claim 23 wherein the poultry are less than 104 weeks ofage.
 27. The method according to claim 26 wherein the poultry are 3weeks of age or less.
 28. The method according to claim 27 wherein thepoultry are less than one day of age.
 29. The method according to claim26 wherein the poultry are chickens.
 30. The method according to claim26 wherein the spray administration is followed by administration of atleast one booster dose of the vaccine.
 31. The method according to claim30 wherein the booster dose of vaccine is administered by sprayadministration.
 32. The method according to claim 30 wherein the boosterdose of vaccine is administered in drinking water.
 33. The methodaccording to claim 30 wherein a booster dose is administered 14 daysafter the spray administration.
 34. A domestic bird that has beentreated by the method of claim 1.